CN102534027A - Detection primer for QacE delta1 genes in MDRPA (multidrug-resistant pseudomonas aeruginosa) and detection method thereof - Google Patents

Abstract

The invention discloses a detection primer for QacE delta1 genes in MDRPA (multidrug-resistant pseudomonas aeruginosa) and a detection method thereof. The detection primer for QacE delta1 genes in MDRPA is characterized by comprising a forward primer 5'-cttccgccgttgtcataatc-3' for QacE delta1 genes and a reverse primer 5' atcaagcttttgcccatgaa-3' for QacE delta1 genes. The detection method comprises the following steps: with a positive clinical sample isolate (which is cultured and identified to be MDRPA positive) as a detecting sample and a bacterial strain of pseudomonas aeruginosa without carrying QacE delta1 genes as a negative reference sample, carrying out high temperature inactivation and extracting the DNA (deoxyribonucleic acid) of the sample; artificially synthesizing a QacE delta1 sequence, and constructing recombinant plasmids (carrying QacE delta1 genes) to be a positive reference sample; and respectively carrying out PCR (polymerase chain reaction). The primer and method disclosed by the invention are high in detection rate and strong in repeatability.

Description

The detection primer and the detection method thereof of QacE Δ 1 gene among the MDRPA

Technical field

The present invention relates to the detection method that the QacE of anti-sterilizing agent Δ 1 gene in a kind of multidrug resistance Pseudomonas aeruginosa (MDRPA) carries situation, belong to technical field of molecular biology.

Background technology

Application and the development existing several centuries of disinfection preservative in life.Come potable water storage from the container made from copper and silver empirically; Clean a wound with vinegar and honey, preserve fish and meat, arrive with the tincture of iodine as the wound disinfection agent with spices; In obstetrics, use chlorinated water solution; As being used as sporicide as antiseptic-germicide with dimercurion in wound dressings and the orthopaedic surgical operations, whole disinfection preservative all is among the continuous development with phenol.The 20 beginning of the century mankind further developed again biguanides (CRAs) and quaternary ammonium sterilizing agent (quatemaryammon ium compounds, QACs).To the forties in 20th century, disinfection preservative commonly used has phenolic cpd, organic mercury, biguanides sterilizing agent, quaternary ammonium sterilizing agent, iodine and mixture thereof, ethanol, formaldehyde, hydrogen peroxide, silver compound, dyestuff (like acridine, tritane) and amphoterics.Up at present, the great majority in these sterilizing agents are still using widely.Among although disinfection preservative is updating always, widely under the selective pressure, bacterium has still produced resistance gradually at it.Have now found that bacterium is relevant with the drug resistant gene of bacterium itself to the resistance of disinfection preservatives such as quaternary ammonium, biguanides and heavy metal.

Separate the I class integron of QacE Δ 1 from plasmid R751 and obtain.This plasmid separates from Klebsiella Pneumoniae at first.Nucleic acid sequencing analysis revealed, 3 of its DNA integrase gene ' end have 3 opening code-reading frames (ORF1, ORF4 and ORF5), and 100 of ORF1 coding have 94 identical with ORF4.It and QacC have high homology.Paulsen etc. are ORF1 called after qacE, ORF4 called after QacE Δ 1, and similar with QacC from phenotype resistance analysis qacE to the resistance spectrum of sterilizing agent, QacE Δ 1 is the resistance of lower level to these sterilizing agents.Therefore, QacE Δ 1 possibly be to be inserted among the ORF1 and to evolve owing to carrying sulfanilamide (SN) (sulI) drug resistant gene dna segment, and it finally causes encoded protein to lack 162 amino acid whose C-terminals than the ORF1 BSA.Fluorescent test shows that the resistance of QacE also is the proton pump mediation.The amino acid sequencing result shows that QacE also belongs to little multidrug resistance family albumen, and four transmembrane segments are arranged.Both are all to quaternary ammonium disinfectant (fragrant like Morpan BB, benzalkonium chloride and bromination rice), biguanide compound (like chlorhexidine), hydrazone compounds and dyestuff resistance.The resistance level of 1 pair of quaternary ammonium disinfectant of QacE Δ and dyestuff is lower than QacE.Further research shows, compares with QacE, and these differences are because QacE Δ 1 albumen has lost the high conservative residue of the 4th transmembrane segment and C-terminal causes.

Detect at present method that QacE Δ 1 gene carries situation and mainly contain agarose gel electrophoresis behind the PCR, order-checking.The gel electrophoresis cost is low, easy to operate, but sensitivity is low, occurs false negative easily; Order-checking is the gold standard that detects transgenation, but sequencing technologies complex steps, length consuming time, pollutes easily.

Summary of the invention

The purpose of this invention is to provide the PCR that the QacE of anti-sterilizing agent Δ 1 gene in the multidrug resistance Pseudomonas aeruginosa (MDRPA) carries situation and detect primer and detection method thereof.

In order to achieve the above object, the invention provides the detection primer of QacE Δ 1 gene among one group of MDRPA, it is characterized in that, comprising:

(A) .QacE Δ 1 gene forward primer: 5 '-cttccgccgttgtcataatc-3 ';

(B) .QacE Δ 1 gene reverse primer: 5 '-atcaagcttttgcccatgaa-3 '.

The present invention also provides the detection method of QacE Δ 1 gene among a kind of MDRPA, it is characterized in that, adopts the detection primer of QacE Δ 1 gene among the above-mentioned MDRPA, and concrete steps are:

The first step: be accredited as MDRPA male clinical sample strain isolated after the cultivation of learning from else's experience, high-temperature inactivation extracts sample DNA, as test sample; Get the pseudomonas aeruginosa strains that does not carry QacE Δ 1 gene, high-temperature inactivation extracts sample DNA, as the negative control article; Synthetic QacE Δ 1 sequence makes up the positive reference substance of recombinant plasmid that carries QacE Δ 1 gene;

Second step: use the QacE Δ 1 gene forward primer solution of sterilized water compound concentration as 5-15umol/L, concentration is the QacE Δ 1 gene reverse primer solution of 5-15umol/L and the QacE Δ 1 gene test probe solution that concentration is 5-15umol/L; The sequence of QacE Δ 1 gene forward primer is: 5 '-cttccgccgttgtcataatc-3 '; The sequence of QacE Δ 1 gene reverse primer is: 5 '-atcaagcttttgcccatgaa-3 ', the sequence of QacE Δ 1 gene test probe is: FAM-tggtcgggactcggc-MGBNFQ;

The 3rd step: add PCR Master Mix 10-15ul and second at each PCR reacting hole in successively and go on foot QacE Δ 1 gene forward primer solution 0.1-1ul, QacE Δ 1 gene reverse primer solution 0.1-1ul, the QacE Δ 1 gene test probe solution 0.1-1ul that obtains; In different PCR reacting holes, add test sample, negative control article and the positive reference substance 0.1-1ul that the first step obtains then respectively, supply 25ul with the sterilization double distilled water; On quantitative real time PCR Instrument, react, reaction conditions is 40-60 ℃ of insulation 1-3 minute, and 80-100 ℃ is incubated 1-3 minute, and 30-50 the circulation of reruning, each circulation are included in 80-100 ℃ and are incubated 10-20 second, again 50-70 ℃ of insulation 0.5-1.5 minute;

The 4th step: carry out interpretation of result with software SDS2.2, the S-shaped curve of PCR result is and carries QacE Δ 1 gene.

The present invention is to whether also having the detection of the QacE of anti-sterilizing agent Δ 1 gene in the multidrug resistance Pseudomonas aeruginosa; In a single day bacterium obtains this gene can be to used sterilizing agent tolerance; Sterilizing agent was lost efficacy,, help monitoring the bacterial strain that contains the gene of anti-the sterilizing agent through detecting the situation of carrying of understanding QacE Δ 1 gene in the bacterium; It is popular to prevent that bacterial strain from continuing, and from the scientific research angle the further understanding of its resistance mechanism is also helped the development of new sterilization preparation simultaneously.

The present invention is based on the real-time fluorescence quantitative PCR technology, use the Taqman-MGB probe of characteristics such as having resolving power is higher, the hybridization specificity is stronger, favorable reproducibility, not only verification and measurement ratio is high, repeatable strong; Detection speed is fast, and total overall reaction is accomplished in the reaction tubes of sealing, has effectively avoided crossed contamination; And level of automation is high; Can monitor in real time, the quality control standard article are provided, make interpretation as a result more directly perceived.

Description of drawings

Fig. 1 is the Realtime PCR graphic representation that carries the positive reference substance of QacE Δ 1 gene;

Fig. 2 is the Realtime PCR graphic representation of Realtime PCR negative control article;

Fig. 3 is the Realtime PCR graphic representation that carries QacE Δ 1 gene test sample;

Fig. 4 is the agarose gel electrophoresis figure of QacE Δ 1 gene test sample;

Fig. 5 is the sequencer map of QacE Δ 1 gene test sample.

Embodiment

Below in conjunction with accompanying drawing and embodiment the present invention is described further.

Embodiment

1, adopt the solid phase phosphoramidite triester method to prepare one group and be used for detecting MDRPA QacE Δ 1 gene and carry the primer of situation, comprising:

QacE Δ 1 gene forward primer: 5 '-cttccgccgttgtcataatc-3 ';

QacE Δ 1 gene reverse primer: 5 '-atcaagcttttgcccatgaa-3 '.

2, detection method:

(1) learns from else's experience and be accredited as multidrug resistance Pseudomonas aeruginosa male clinical sample strain isolated after cultivating; High-temperature inactivation; (Yi Rui Bioisystech Co., Ltd in Shenzhen YP03002) extracts sample DNA, is diluted to 30ul (concentration 10ng/ul) to extract test kit with commercial bacterial genomes DNA magnetic bead; Put-20 ℃ of preservations, for use; With standard Pseudomonas aeruginosa strain (depositary institution: Chinese agriculture microbial strains preservation administrative center; Deposit number:

high-temperature inactivation; Extract test kit (Shenzhen Yi Rui Bioisystech Co., Ltd with commercial bacterial genomes DNA magnetic bead; YP03002) extract sample DNA; Be diluted to 30ul (concentration 10ng/ul), as the negative control article; Synthetic QacE Δ 1 sequence (it is synthetic to give birth to the worker by Shanghai) makes up the recombinant plasmid that carries QacE Δ 1 gene, is diluted to 30ul (concentration 10ng/ul), positive reference substance.

(2) according to QacE Δ 1 gene conservative zone; Adopt ABI Primer Express 3.0 real-time fluorescence quantitative PCR primer-design softwares; Design synthetic primer and Taqman probe, probe sequence one end is marked with the report optical dye, and the other end is marked with cancellation optical dye (it is synthetic to give birth to the worker by Shanghai); Use the QacE Δ 1 gene forward primer solution of sterilized water compound concentration as 10umol/L; Concentration is the QacE Δ 1 gene reverse primer solution of 10umol/L and the QacE Δ 1 gene test probe solution that concentration is 10umol/L, and wherein, primer and detection probes sequence are following:

QacE Δ 1 gene forward primer: 5 '-cttccgccgttgtcataatc-3 ';

QacE Δ 1 gene reverse primer: 5 '-atcaagcttttgcccatgaa-3 ';

QacE Δ 1 gene test probe: FAM-tggtcgggactcggc-MGBNFQ.

(3) in each PCR reacting hole, add PCR Master Mix (the Taqman Universal PCR Master Mix 2X that Applied Biosystems company produces; Comprise 10 * PCR damping fluid, MgC12, dNTP, archaeal dna polymerase) 12.5ul; QacE Δ 1 gene forward primer solution 0.5ul; QacE Δ 1 gene reverse primer solution 0.5ul, QacE Δ 1 gene test probe solution 0.5ul.In different PCR reacting holes, add test sample, negative control article or the positive reference substance 0.5ul that step (1) obtains then respectively, supply 25ul with the sterilization double distilled water.On ABI7300 type quantitative real time PCR Instrument, react, reaction conditions is 50 ℃ of insulations 2 minutes, and 95 ℃ are incubated 2 minutes, rerun 40 and circulate, and each circulation is included in 95 ℃ of insulations 15 seconds, again 60 ℃ of insulations 1 minute;

(4) carry out interpretation of result with software SDS2.2, as shown in Figure 1, the Realtime PCR curve of positive reference substance, S-shaped.As shown in Figure 2, the Realtime PCR curve of negative reference substance is non-S shape.As shown in Figure 3, for the Realtime PCR curve of test sample, S-shaped.Draw judgement, this sample is for carrying QacE Δ 1 gene bacterial strain.

Utilize the present invention that the detected result that surpasses 100 routine MDRPA samples is shown, QacE Δ 1 gene that carries out according to aforesaid method carries the situation recall rate and can reach more than 99%, and repeatability reaches 100%.And the recall rate of agarose gel electrophoresis method is about 75%, and is as shown in Figure 4, is the agarose gel electrophoresis figure of QacE Δ 1 gene test sample; The present invention compares with direct sequencing, and consistence can reach more than 99.5% as a result, and is as shown in Figure 5, is the sequencer map of QacE Δ 1 gene test sample.

Claims (2)

1.MDRPA the detection primer of middle QacE Δ 1 gene is characterized in that, comprising:

(A) .QacE Δ 1 gene forward primer: 5 '-cttccgccgttgtcataatc-3 ';

(B) .QacE Δ 1 gene reverse primer: 5 '-atcaagcttttgcccatgaa-3 '.

2. the detection method of QacE Δ 1 gene among the MDRPA is characterized in that, adopts the detection primer of QacE Δ 1 gene among the described MDRPA of claim 1, and concrete steps are:

The first step: be accredited as MDRPA male clinical sample strain isolated after the cultivation of learning from else's experience, high-temperature inactivation extracts sample DNA, as test sample; Get the pseudomonas aeruginosa strains that does not carry QacE Δ 1 gene, high-temperature inactivation extracts sample DNA, as the negative control article; Synthetic QacE Δ 1 sequence makes up the positive reference substance of recombinant plasmid that carries QacE Δ 1 gene;

Second step: use the QacE Δ 1 gene forward primer solution of sterilized water compound concentration as 5-15umol/L, concentration is the QacE Δ 1 gene reverse primer solution of 5-15umol/L and the QacE Δ 1 gene test probe solution that concentration is 5-15umol/L; The sequence of QacE Δ 1 gene forward primer is: 5 '-cttccgccgttgtcataatc-3 '; The sequence of QacE Δ 1 gene reverse primer is: 5 '-atcaagcttttgcccatgaa-3 ', the sequence of QacE Δ 1 gene test probe is: FAM-tggtcgggactcggc-MGBNFQ;

The 3rd step: add PCR Master Mix 10-15ul and second at each PCR reacting hole in successively and go on foot QacE Δ 1 gene forward primer solution 0.1-1ul, QacE Δ 1 gene reverse primer solution 0.1-1ul, the QacE Δ 1 gene test probe solution 0.1-1ul that obtains; In different PCR reacting holes, add test sample, negative control article and the positive reference substance 0.1-1ul that the first step obtains then respectively, supply 25ul with the sterilization double distilled water; On quantitative real time PCR Instrument, react, reaction conditions is 40-60 ℃ of insulation 1-3 minute, and 80-100 ℃ is incubated 1-3 minute, and 30-50 the circulation of reruning, each circulation are included in 80-100 ℃ and are incubated 10-20 second, again 50-70 ℃ of insulation 0.5-1.5 minute;

The 4th step: carry out interpretation of result with software SDS2.2, the S-shaped curve of PCR result is and carries QacE Δ 1 gene.

Images